1. Field of the Invention
The present invention provides methods, compositions and kits related to making double-tagged DNA libraries using a double-tagged oligonucleotide. It particularly relates to methods, compositions and kits for making di-tagged dsDNA libraries suitable for high throughput sequencing from DNA and RNA samples.
2. Description of the Related Art
Next Generation Sequencing (NGS) is a high-throughput sequencing technology that performs thousands or millions of sequencing in parallel. The NGS technology enables researchers to answer fundamental biological questions at a genomic scale and has great potential in medical applications. The first step to apply NGS technologies is constructing DNA libraries of random DNA fragments generated from DNA/RNA samples, having different sequence tags attached at both ends. Preparation of high quality di-tagged DNA library is critical for successful sequencing.
As Next Generation Sequencing technologies evolve and advance, there are growing demands for PCR free library preparation, Mate Pair library preparation and single stranded library preparation to serve researchers with specific project needs. The challenges of PCR free library preparation is the ligation step: current method involves ligating two different sequence tags to blunt ended DNA fragments using a DNA ligase. Due to random ligation of different tags to ends of DNA fragments, ligation products include DNA sequences with only one tag, with the same tags, or with two different tags. PCR amplification is needed to selectively amplify the DNA fragments having different tags. An improved ligation method uses one “Y” shaped tag having a non-complementary outer portion and a complementary inner portion. Two strands of the non-complementary outer portion have different sequence tags, and two strands of the complementary inner portion anneal to each other to form a dsDNA that can be ligated to dsDNA fragments. Once a DNA is ligated to two “Y” shaped tags, each DNA strand will be ligated to different sequence tags of the non-complementary outer portion. However, selective PCR amplification is still needed since this method does not exclude the possibility that DNA is ligated to only one “Y” shaped tag.
Using a double-tagged oligonucleotide, the present invention provides a method of making a di-tagged DNA without the need of performing a PCR, therefore eliminating the sequence bias caused by PCR.
A mate pair DNA refers to a DNA sequence comprising two DNA segments originally located long distance apart in the genome. A mate pair library is comprised of mate pair DNA sequences with two different sequence tags attached at the ends. Mate pair sequencing is useful for many applications such as genomic sequence assembly, assessment of genomic rearrangement, and assembly of repetitive sequences. To make a mate pair library, end sequences of a large DNA fragment are connected together and attached to two different sequence tags. Current techniques for preparing mate pair DNA libraries involve fragmenting genomic DNA into large fragments, performing end repairs, labeling ends of large DNA fragments with biotin, self-ligation of biotinylated ends, random fragmentation to generate smaller DNA fragments, isolation of biotinylated fragments, performing end repairs, and ligation of two different tags to the DNA fragments. This procedure comprises many enzymatic and cleaning steps leading to low yield of mate pair DNAs. The low ligation efficiency of biotinylated nucleotides further limits the application of this method.
Using a double-tagged oligonucleotide, the present invention provides a method of making Mate-Pair DNA libraries with less enzyme reactions and less purification steps, resulting in simpler workflow and better recovery.
Single stranded DNA library has many applications such as DNA methylation analysis and hybridization based target capture. Existing method for generating single stranded DNA involves ligation of an adaptor to a dsDNA molecule, and uses only one primer to linearly amplify one single strand. One of the major drawbacks for this method is the sequence bias introduced by PCR cycles.
Using a double-tagged oligonucleotide, the present invention provides a simple PCR-free method to generate single stranded DNA library from dsDNA, eliminating the sequence bias caused by PCR. The resulting library would well reflect the complexity and composition of the DNA sequences of the starting materials.
RNA sequencing (RNA Seq) uses high throughput sequencing technology for quantifying and mapping transcriptomes, enabling rapid profiling and deep investigation of transcriptional activities. The first step of RNASeq is to convert a population of RNA molecules into a library of DNA molecules with different tags attached to both ends, which can then be sequenced in a high throughput manner. The conventional method for preparing tagged DNAs from RNAs involves reverse transcription to generate first and second complementary DNA (cDNA) strands, DNA fragmentation, DNA ends repairing, adaptor ligation, and PCR amplification. These procedures include multiple enzyme reactions and buffer exchanges between different enzyme reactions, resulting in significant loss of starting materials. Sequence-specific bias may also be introduced during ligation and amplification steps.
Using a single stranded, double-tagged oligonucleotide, the present invention provides a method of making RNA libraries without synthesizing second strand cDNA, resulting not only simpler work flow, but also provides directional information on the RNA Transcripts. More importantly, it offers a library preparation solution to RNA samples of extremely small amounts, such as applications for CLIP Seq.